We propose to continue our project to investigate the mechanism of RNA localization. Over the past years we have shown the beta-actin mRNA contains sequence information which specifies cellular spatial location. This occurs in fibroblasts and neurons where beta-actin is localized to the leading edge of the lamella or the growth cone and appears to be important for regulation of actin polymerization and hence cell polarity and motility. We have identified the cis-acting sequences and at least two trans-acting factors. Interestingly these elements and factors are identical in neurons and fibroblasts. We have identified the proteins which bind specifically to the RNA 'zipcodes'. We hypothesize that they link the RNA to regulatory factors. For fibroblasts and neurons, one of these proteins appears to be ZBP1 (zipcode binding protein 1), a KH domain hnRNP which shuttles between the nucleus and cytoplasm. The nuclear and cytoplasmic functions of this protein may indicate that an RNA is 'marked' in the nucleus for its localization before export to the cytoplasm and subsequent assembly with transport machinery. In fact, we show that ZBP1 binds to beta-actin pre-mRNA as it is being transcribed. There are other proteins associated with the beta-actin mRNA which may form a complex with ZBP1, one of which is ZBP2, homologous to a splicing factor, KSRP. We hypothesize that this complex is responsible for the localization of the mRNA, and can travel either by microtubule- or actin-based motors. We visualized this ZBP1-RNA complex as a particle (the "locasome"). This proposal proposes to continue to characterize this ZPB1-RNA complex biochemically, molecularly and cell biologically for its role in mRNA localization. The sorting of this complex requires cytoskeletal elements and motors. In fibroblasts an actomyosin motor system appears to be important. Deletion of the gene for myosin IIB stops all localization in fibroblasts. However, in neurons, a microtubule-based motor appears to control the localization in dendrites and axonal processes. Once the complex is localized, we hypothesize that factors involved in RNA translation become operable. One of these is EF1alpha, a translation factor and actin binding protein that also interacts with ZBP1. We will investigate the pathway using high-resolution microscopy combined with FRET to visualize the spatial distribution of these protein interactions in real time.